Project Summary Campylobacter jejuni [unreadable] [unreadable]a NIAID Category B Priority Pathogen[unreadable] [unreadable] is a leading bacterial cause of human gastrointestinal disease worldwide. While the clinical presentation of C. jejuni infection is similar to Salmonella typhimurium, the virulence determinants and mechanisms that C. jejuni use to cause disease are unique. Our ultimate goal is to reduce the incidence and severity of C. jejuni infection. To accomplish this goal, we need a better understanding of the fundamental mechanisms of C. jejuni pathogenesis. We discovered that C. jejuni synthesizes at least two Microbial Surface Components Recognizing Adhesive Matrix Molecule(s) (MSCRAMM), designated CadF and FlpA, that promote the bacterium[unreadable]s binding to fibronectin. We have also discovered that maximal cell invasion requires the Campylobacter invasion antigens (Cia). The Cia proteins are defined as the proteins exported from the bacterium[unreadable]s flagellar Type III Secretion System (T3SS) that presumably modify host cell cytoskeletal regulatory proteins to promote C. jejuni-host cell entry and intracellular survival. However, significant gaps exist in our knowledge of the molecular mechanisms of C. jejuni binding and internalization. Based on our data and published papers, we hypothesize: 1) C. jejuni binds to fibronectin and stimulates [unreadable]outside-in[unreadable] signaling in epithelial cells that sets the stage for invasion by recruiting structural and signaling molecules to the sites of bacterial attachment;2) C. jejuni Cia proteins exported from the flagellar export apparatus are delivered to the cytoplasm of host cells;and 3) C. jejuni Cia proteins stabilize or modify cellular structural components and signaling pathways to promote host cell entry and intracellular survival. The novelty of this proposal is that C. jejuni binding and invasion factors work cooperatively and simultaneously to modify host cell pathways, which when dissected completely will offer multiple targets for therapeutic intervention. The experiments outlined in this proposal are possible because we have the expertise to perform a variety of in vitro and in vivo assays and well-characterized C. jejuni mutants deficient in binding (e.g., cadF and flpA mutants), invasion (e.g., ciaB and ciaC mutants), and host cell survival (e.g., ciaI mutant). This study will result in the identification of novel C. jejuni virulence factors, and how these factors alter host cell signaling pathways to promote host cell invasion.